1. Field of the Invention
This invention relates to matrix metalloproteinase enzymes, inhibitors of matrix metalloproteinase enzymes, and to methods of changing the conformation of matrix metalloproteinase enzymes.
2. Description of Related Art
Connective tissue, extracellular matrix constituents, and basement membranes are required components of all mammals, including humans. These components are the biological materials that provide rigidity, differentiation, attachments, and, in come cases, elasticity to biological systems. Connective tissue components include, for example, collagen, elastin, proteoglycans, fibronectin, and laminin. These biochemicals make up or are components of structures such as skin, bone, teeth, tendons, cartilage, basement membranes, blood vessels, cornea, and vitreous humor.
Under normal conditions, connective tissue turnover or repair processes are controlled and in equilibrium. The loss of this balance for whatever reason leads to a number of disease states. Inhibition of the enzymes responsible for loss of equilibrium provides a control mechanism for this tissue decomposition and, therefore, a treatment for these diseases.
Degradation of connective tissue or connective tissue components is carried out by the action of proteinase enzymes released from resident tissue cells or invading inflammatory or tumor cells. A major class of enzymes involved in this function includes the matrix metalloproteinase (MMP) enzymes. The MMPs are the subject of extensive study because of their potential involvement in disease mechanisms. Parks and Mecham have extensively reviewed the MMPs (Matrix Metalloproteinases, W. C. Parks and R. P. Mecham, ed., Academic Press, San Diego (1998)).
The MMPs are divided into classes with some members having several different names in common use. Examples are: collagenase I (MMP-1, fibroblast collagenase, EC 3.4.24.3); collagenase II (MMP-8, neutrophil collagenase, EC 3.4.24.34); collagenase III (MMP-13); stromelysin 1 (MMP-3, EC 3.4.24.17); stromelysin 2 (MMP-10, EC 3.4.24.22); proteoglycanase; matrilysin (MMP-7, EC 3.4.25.33); gelatinase A (MMP-2, 72 kDa gelatinase, EC 3.4.24.24); gelatinase B (MMP-9, 92 kDa gelatinase, EC 3.4.24.35); stromelysin 3 (MMP-11); metalloelastase (MMP-12, HME, human macrophage elastase, EC 3.4.24.65); MT1-MMP (MMP-14); MT2-MMP (MMP-15); MT3-MMP (MMP-16); and MT4-MMP (MMP-17).
The uncontrolled breakdown of connective tissue by MMPs is a feature of many pathological conditions. Examples include rheumatoid arthritis, osteoarthritis, septic arthritis, ulcerations (such as corneal, epidermal, or gastric ulcerations), periodontal disease, proteinuria; Alzheimer's Disease, coronary thrombosis, psoriasis, aneurysm, and bone disease. Defective injury repair processes also occur. This can produce improper wound healing leading to weak repairs, adhesions, and scarring. These latter defects can lead to disfigurement and/or permanent disabilities as with post-surgical adhesions.
MMP-8 (also known as neutrophil collagenase) has been shown to degrade type II collagen and aggrecan (a structural glycosaminoglycan found in the cartilage). MMP-8 has been found to be present in patients having osteoarthritis and rheumatoid arthritis and may participate significantly in the progression of these diseases. Matrix Metalloproteinases, W. C. Parks and R. P. Mecham, ed., Academic Press, San Diego (1998), pp. 32-33.
MMPs are also involved in the biosynthesis of tumor necrosis factor (TNF). Inhibition of the production or action of TNF and related compounds is a useful clinical disease treatment mechanism. TNF-α, for example, is a cytokine that is believed to be produced initially as a 28 kDa cell-associated molecule. It is released as an active, 17 kDa form that can mediate many deleterious effects in vitro and in vivo. For example, TNF can cause or contribute to the effects of inflammation, rheumatoid arthritis, autoimmune disease, multiple sclerosis, graft rejection, fibrotic disease, cancer, infectious diseases, malaria, mycobacterial infection, meningitis, fever, psoriasis, cardiovascular/pulmonary effects (such as post-ischemic reperfusion injury, congestive heart failure hemorrhage, coagulation, and hyperoxic alveolar injury), radiation damage, and acute phase responses like those seen with infections and sepsis during shock such as septic shock and hemodynamic shock. Chronic disease of active TNF can cause cachexia and anorexia. Chronic release of TNF can be lethal.
TNF-α convertase is a metalloproteinase involved in the formation of active TNFα. Inhibition of TNF-α convertase inhibits production of active TNF- α. Some compounds that inhibit TNF-α convertase and MMPs involved in TNF-α biosynthesis are disclosed in PCT Patent Application No. WO 94/24140. Additional compounds that inhibit such enzymes are disclosed in PCT Patent Application No. WO 94/02466. Further inhibitors are disclosed in PCT Patent Application No. WO 97/20824.
Some compounds that inhibit certain MMPs have been shown to also inhibit the release of TNF (Gearing et al., Nature, 376, 555-557 (1994)). McGeehan et al. disclosed further compounds which inhibit MMPs and inhibit the release of TNF (Nature, 376, 558-561 (1994)). There remains a need for effective MMP and TNF-α convertase-inhibiting agents.
MMPs are involved in other biochemical processes as well. Included are the control of ovulation, post-partum uterine involution, possibly implantation of fertilized ova, cleavage of APP (β-Amyloid Precursor Protein) to the amyloid plaque and inactivation of α1-protease inhibitor (α1-PI). Inhibition of these Metalloproteinases permits, for example, the control of fertility and the treatment or prevention of Alzheimer's Disease. In addition, increasing and maintaining the levels of an endogenous or administered serine protease inhibitor drug or biochemical such as α1-PI supports the treatment and prevention of diseases such as emphysema, pulmonary diseases, inflammatory diseases, and diseases of aging such as loss of skin or organ stretch and resiliency.
Inhibition of selected MMPs can also be desirable in other instances. For example, selective inhibition of MMP-3, MMP-2, MMP-9, or MMP-13 in the presence of MMP-1 may be useful for the treatment of cancer, prevention of metastasis of cancer cells, or the inhibition of angiogenesis. A therapy which does not inhibit MMP-1 but does selectively inhibit one or more of the other MMPs can have a therapeutically useful profile.
Osteoarthritis, another prevalent disease wherein it is believed that cartilage degradation in flamed joints is at least partially caused by MMP-13 released from cells such as stimulated chrondrocytes, may be best treated by administration of drugs which selectively inhibit MMP-13. See, for example, Mitchell et al., J. Clin. Invest., 97, 761-768 (1996). See also Reboul et al., J. Clin. Invest., 97, 2011-2019 (1996).
Some inhibitors of MMPs are known. Examples include natural biochemicals such as tissue inhibitor of metalloproteinase (TIMP), a2-macroglobulin, and their analogs or derivatives. These are high molecular weight protein molecules that form inactive complexes with Metalloproteinases.
Some smaller peptide-like compounds that inhibit MMPs have also been described. Thiol group-containing amide or peptidyl amide-based metalloproteinase (MMP) inhibitors are known as is shown in, for example, PCT Patent Application No. WO 95/12389. Further such inhibitors are described in PCT Patent Application No. WO 97/24117. Still further such inhibitors are shown in U.S. Pat. No. 4,595,700. Hydroxamate group-containing MMP inhibitors are disclosed in a number of individual patent applications such as each of the following:
WO 95/29892.
WO 97/24117.
EP 0 780 386.
WO 90/05719.
WO 93/20047.
WO 95/09841.
WO 96/06074.
Swartz et al. disclose some peptidomimetic MMP inhibitors in Progr. Med. Chem., 29, 271-334 (1992). Further peptidomimetic MMP inhibitors are disclosed by Rasmussen et al., in Pharmacol. Ther., 75(1), 69-75 (1997). Denis et al., disclose further peptidomimetic MMP inhibitors in Invest. New Drugs, 15(3), 175-185 (1997).
One possible problem associated with many known MMP inhibitors is that they often exhibit the same or similar inhibitory effects against each of the MMP enzymes. In other words, many known MMP inhibitors are not very selective. For example, the peptidomimetic hydroxamate known as batimastat is reported to exhibit IC50 values of about 1 to about 20 nanomolar (nM) against each of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9. Marimastat, another peptidomimetic hydroxamate, was reported to be another broad-spectrum MMP inhibitor with an enzyme inhibitory spectrum similar to batimastat, except that marimastat exhibits an IC50 value against MMP-3 of about 230 nM. (Rasmussen et al., Pharmacol. Ther., 75(1), 69-75 (1997))
Meta analysis of data from Phase I/II studies using marimastat in patients with advanced, rapidly progressive, treatment-refractory solid tumor cancers (colorectal, pancreatic, ovarian, prostate) indicated a dose-related reduction in the rise of cancer-specific antigens used as surrogate markers for biological activity. The most common drug-related toxicity of marimastat in those clinical trials was musculoskeletal pain and stiffness, often commencing in the small joints and the hands, spreading to the arms and shoulder. A short dosing holiday of 1-3 weeks followed by dosage reduction permitted treatment to continue. (Rasmussen et al., Pharmacol. Ther., 75(1), 69-75 (1997))It is thought that the lack of specificity of inhibitory effect among the MMPs may be the cause of that effect.
The primary, secondary, and tertiary structures of the MMPs have a number of characteristic features. Each MMP contains a catalytic domain which in turn comprises a zinc binding site and an adjacent site known as the S1′ pocket. The S1′ pocket has been recognized as a major factor in substrate specificity of the MMPs. See for example B. Lovejoy et al., Nat. Struct. Biol., 6 (3), 217-221 (1999) at 218. The S1′ pocket is sometimes known as the specificity pocket.
FIG. 1 shows a partial sequence alignment for MMP-1, MMP-3, and MMP-8. The amino acid residues in the shaded boxes of FIG. 1 comprise the residues included in the S1′ pocket for each of these MMPs. Symbols in FIG. 1 identifying the amino acid residues are commonly used by those of skill in the art. The primary, secondary, and tertiary structures work together for each MMP to provide the catalytic activity, kinetics, and substrate specificity of the enzyme. These structures define the shape or conformation of the amino acid residue backbone of the enzyme. The actual sequence of amino acid residues in the S1′ pocket and the conformation of the residue backbone determine the specificity and kinetics of each MMP.
Some X-ray crystallographic experiments on MMPs are reported in the literature. For example, F. Grams et al. (Euro J. Biochem., 228, 830-841 (1995)) discloses X-ray structures of human neutrophil collagenase complexed with peptidomimetic hydroxamate and thiol inhibitors. In another report B. Lovejoy et al. (Nat. Struct. Biol., 6 (3), 217-221 (1999)) disclose X-ray crystal structures of the catalytic domains of MMP-1 and MMP-13. Lovejoy et al. report that the MMP-1 S1′ pocket undergoes a conformational change to accommodate certain diphenylether inhibitors but that the MMP-13 S1′ pocket is larger and can accommodate the diphenylether inhibitors without undergoing a conformational change. They report that this difference determines the selectivity of these diphenylether compounds for preferentially inhibiting MMP-13 relative to MMP-1. The X-ray crystal structure for MMP-2 was reported by E. Morgunova et al. (“Structure of Human Pro-Matrix Metalloproteinase-2: Activation Mechanism Revealed,” Science 284, 1667-1670 (1999)).